Plasma Factor XIII is an essential molecule for normal hemostasis in man. When deficiencies of Factor XIII occur or inhibitors are present they are always associated with severe, life-threatening, bleeding. This provides strong evidence that Factor XIII plays a vital role in maintaining the stability of blood clots, or the "hemostatic plug."
Plasma Factor XIII is composed of two a-chains and two b-chains which are noncovalently associated. See generally J. McDonagh, in Hemostasis and Thrombosis, 289-300 (R. Colman et al. eds. 1987). Conversion of plasma Factor XIII to its active transglutaminase form, Factor XIIIa, occurs in two steps. In the first step, thrombin cleaves a 4,000 dalton peptide from each of the a-chains with the formation of an inactive intermediate (a.sub.2 b.sub.2). T. Takagi and R. Doolittle, 13 Biochemistry 750 (1974). In the next step, calcium causes the b-chains to dissociate and the active site cysteines on the thrombin-cleaved a-chains are exposed, producing Factor XIIIa (a.sub.2). C. Curtis et al., 13 Biochemistry 13774 (1974).
During blood clotting, Factor XIIIa catalyzes the formation of intermolecular .tau.-glutamyl-.epsilon.-lysyl bonds between .tau.-chains and .epsilon.-chains of polymerizing fibrin monomers. Covalently modified fibrin is mechanically stronger and more resistant to lysis by plasmin when compared to noncrosslinked fibrin. See L. Shen and L. Lorand, 71 J. Clin. Invest. 1336 (1983); Y. Sakata and N. Aoki, 65 J. Clin. Invest. 290 (1980).
The complete amino acid sequence of the a-chain was published based on protein sequence analysis and analysis of the cDNA sequence. See N. Takahashi et al., 83 Proc. Natl. Acad. Sci. USA 8019 (1986); U. Grundman et al., 83 Proc. Natl. Acad. Sci. USA 8024 (1986); A. Ichinose et al., 25 Biochemistry 6900 (1986). The entire intron and exon structure of the Factor XIII a-chain gene was recently published. A. Ichinose et al., 85 Proc. Natl. Acad. Sci. USA 5829 (1988). While little is known about how the structure of this protein relates to its function, we have found that cleavage of the activation and inactivation peptides releases a monomeric 51 kD fibrin binding peptide. See C. Greenberg et al., 256 Biochem. J. 1013 (1988).
The a-chain of plasma Factor XIII has 9 free sulfhydryl groups, one of which is in the active site of Factor XIIIa, and no disulfide bonds. The active site has sequence homology to calpains and guinea pig liver transglutaminase. See K. Ikura et al., 27 Biochemistry 2898. Tissue transglutaminases were responsible for the earliest clotting mechanisms. R. Myherman and J. Bruner-Lorand, 19 Meth. Enzymol. 765 (1970). Guinea pig liver transglutaminase is one of the more extensively studied tissue transglutaminases. Guinea pig liver transglutaminase was recently cloned and was found to share several homologous domains with Factor XIII a-chains. See K. Ikura et al., 27 Biochemistry 2898 (1988). We found that the two proteins shared homologous fibrin binding domains. See K. Achyuthan et al., J. Biol. Chem. 263, 14296 (1988). We detected six homologous regions when we compared the sequence of Factor XIII and the guinea pig liver transglutaminase See K. Achyuthan et al., in Fibrinogen 3: Biochemistry, Biological Functions, Gene Regulation and Expression, 165-69 (M. Mosesson et al. eds. 1988).
The present invention is based on our ongoing research into the structure and function of Factor XIIIa.